Glutathione (GSH) is a biologically active tripeptide compound formed by condensation of L-glutaminic acid, L-cysteine and glycine, and is widely present in animal, plant and microbial cells. GSH has a variety of important physiological functions within organisms, and particularly plays an important role in the maintenance of a suitable oxidation-reduction environment within the organisms, so that it has wide applications in clinical, food and cosmetic industries.
Methods for producing GSH mainly include chemical synthesis methods, enzyme methods, fermentation methods and the like. In the chemical synthesis methods, three precursor amino acids are used as raw materials to carry out chemical synthesis, but it is not easy to separate the resulting active products and thus the product purity is not high, so that the application of these methods is limited. In the enzyme synthesis, three substrate amino acids and GSH within the organisms are used to synthesize related enzymes, and GSH is synthesized by the addition of a small amount of ATP. This method suffers from complex operation and higher cost. In the fermentation methods, low-cost sugars may be used as raw materials, to synthesize GSH through specific microbial metabolism, which is simple in operation, lower in cost, higher in the production rate, and easy to upscale. Therefore, increasing importance is attached to the production of GSH by fermentation methods.
In the microbial world, strains generating GSH mainly concentrate in eukaryotic yeasts and Gram negative bacteria. Wild Saccharomyces cerevisiae and Candida utilis themselves have relatively high GSH contents, and can continuously maintain GSH synthesis capability, and therefore they have become the most common strains in the industrial production of GSH.
Chinese Invention Patent Application with an Application No. 201511003961.9 titled “Saccharomyces cerevisiae with high yield of glutathione and use thereof” discloses Saccharomyces cerevisiae with high yield of glutathione and use thereof. This yeast has a high yield of glutathione after fermentation culture, and has a short fermentation period. In that invention, carbon and nitrogen sources, vitamins, and precursor amino acids are sequentially added respectively in a manner of supplement by sequential addition to promote the synthesis of glutathione, so as to increase the glutathione content within yeast cells, and effectively solve the problems of low glutathione synthesis capability of yeasts and low glutathione contents in dry yeasts.
Though wild Saccharomyces cerevisiae and Candida utilis themselves have higher GSH contents, the yields thereof are only suitable for use in laboratory studies, and cannot satisfy requirements in industrial production. Screening strains with high yields of GSH on specific media through physical or chemical mutagenesis is a main means to improve the yield and performance of strains.
In general, the GSH content in microbial cells is not high, and accounts for only 0.1 to 1% of dry weight of the cells. GSH at a too high content will be easy to impair the oxidation-reduction environment that has been balanced in the cells. GSH is an intracellular product and needs extraction in the practical production process, and a lower content thereof will no doubt greatly increase the production cost. Therefore, how to improve the cell density and intracellular GSH content has become a key issue in the production of GSH through fermentation methods.
Chinese Patent with an Application No. 200810105972.1 titled “a Saccharomyces cerevisiae strain, dry yeast rich in reduced glutathione and preparation method thereof” provides a yeast strain (CCTCC M 205130) with a higher content of reduced glutathione and stable performance screened out through an ultraviolet mutagenesis method. The strain of this invention is directly used together with yeast cells without extracting and purifying glutathione so as to obtain the dry yeast rich in reduced glutathione.
Moreover, many functions of GSH are exhibited mainly by a mercapto group in the molecule, and the mercapto group is provided by L-cysteine in the synthesis process of GSH. Typically, the amount of L-cysteine synthesized in the cells themselves is lower, which also becomes a limiting factor in large-scale synthesis of GSH in cells. Therefore, exogenous addition of L-cysteine is more effective for the improvement in the GSH synthesis rate.
Chinese Invention Patent with a Patent No. ZL03113418.1 titled “Method for improving yield of glutathione produced by fermentation with tornla yeast” discloses a method for producing glutathione by fermentation, including: adopting torula yeast as the fermentation strain, and after slant culture and seed culture, adding L-cysteine into the fermentation culture medium, to increase supply of L-cysteine in the fermentation broth, so as to improve the synthesis rate and yield of glutathione.
However, an excess of L-cysteine will influence the cell growth. No method exists in the prior art yet that can cross this barrier.